One-tube direct detection of double stranded DNA mutations by a mismatch endonuclease I/CRISPR cas12a cascading system

DNA mutations play a critical role in the pathogenesis of diseases, highlighting their importance in disease diagnosis and treatment. However, DNA mutations predominantly manifest as double-stranded DNA (dsDNA), presenting a challenge for current DNA analysis methods that lack the capability to directly detect dsDNA. The conversion of dsDNA into single strand, followed by subsequent purification steps, introduces complexity to the detection process and diminishes sensitivity. To address these limitations, our study presents the design of a Mismatch Endonuclease I (ME I)/CRISPR-Cas12a system that enables direct detection of dsDNA mutations without the need for purification and single-strand conversion. By harnessing the high selectivity of ME I for DNA mutations and the recognition capability of the CRISPR-Cas12a system for sticky-end dsDNA, we have developed a highly sensitive and convenient one-tube assay for dsDNA mutation detection. The system demonstrated a remarkable ability to sensitively detect three specific mismatches (GG, TT, and GT), corresponding to eight distinct mutation types, with discrimination factors ranging from 144 to 310. Moreover, the detection limits for synthetic oligonucleotides and genomic DNA were determined to be 0.1 % and 0.01 %, respectively. Furthermore, we established a simplified workflow for clinical samples and successfully detected the EGFR_T790M mutation in 18 lung cancer samples, with results consistent with NGS. By enabling convenient and sensitive detection of dsDNA mutations, this approach holds substantial clinical significance and broad applicability. •A ME I/CRISPR-Cas12a system enables direct detection of dsDNA mutation is designed.•A One-tube dsDNA mutation assay with detection limits of 0.01 % for genomic DNA.•The system sensitively detected 18 lung cancer samples with EGFR_T790M mutations.